Technology to detect minute quantities of nucleic acids has advanced rapidly over the last two decades including the development of highly sophisticated hybridization assays using probes in amplification techniques such as PCR. Researchers have readily recognized the value of such technology to detect diseases and genetic features in human or animal test specimens. The use of probes and primers in such technology is based upon the concept of complementarity, that is the bonding of two strands of a nucleic acid by hydrogen bonds between complementary nucleotides (also known as nucleotide pairs).
PCR is a significant advance in the art to allow detection of very small concentrations of a targeted nucleic acid. The details of PCR are described, for example, in U.S. Pat. Nos. 4,683,195 (Mullis et al), 4,683,202 (Mullis) and 4,965,188 (Mullis et al), although there is a rapidly expanding volume of literature in this field. Without going into extensive detail, PCR involves hybridizing primers to the strands of a targeted nucleic acid (considered "templates") in the presence of a polymerization agent (such as a DNA polymerase) and deoxyribonucleoside triphosphates under the appropriate conditions. The result is the formation of primer extension products along the templates, the products having added thereto nucleotides which are complementary to the templates.
Once the primer extension products are denatured, one copy of the templates has been prepared, and the cycle of priming, extending and denaturation can be carried out as many times as desired to provide an exponential increase in the amount of nucleic acid which has the same sequence as the target nucleic acid. In effect, the target nucleic acid is duplicated (or "amplified") many times so that it is more easily detected. Despite the broad and rapid use of PCR in a variety of biological and diagnostic fields, there are still practical limitations which must be overcome to achieve the optimum success of the technology. PCR also produces considerable inefficiency in the use of expensive reagents.
Many amplification procedures yield nonspecific side products of nucleic acids that are not targeted. Sometimes nonspecificity is caused by mispriming by the primers whereby they anneal to non-targeted nucleic acids. Many PCR procedures also yield primer dimers or oligomers and double-stranded side products containing the sequences of several primer molecules joined end-to-end. All of these unwanted products adversely affect accurate and sensitive detection of the target nucleic acid.
The problem caused by unwanted side products is particularly acute when the target nucleic acid is present in very low concentrations, for example, less than about 1000 molecules. Such low numbers of molecules can arise from early stages of infectious diseases or because of a very small specimen, such as may be the situation with forensic investigations.
The high sensitivity of PCR makes the process especially susceptible to contamination where amplified target nucleic acid from one reaction is transferred into subsequent reactions using the same primers, generating a false positive in the later reactions.
Under ideal conditions for PCR, the primers used will bind very specifically to the target nucleic acid only, particularly at elevated temperatures used in the process. However, the reaction mixture may also be held at lower temperatures at certain times (for example during manufacture, shipping or before use by a customer), and the primers may undesirably bind to the non-targeted nucleic acids. If this occurs, nonspecific primer extension products and primer dimers can form which can be amplified along with the target nucleic acid during PCR cycles at elevated temperatures. These undesired products can obscure any amplified target nucleic acid (that is, produce high background). The primers are less efficient in amplification of the target nucleic acid, and thus the process requires more of the highly expensive reagents to produce an accurate result. Because reagents in the reaction are utilized to make non-specific products, less specific product is produced, rendering the process less sensitive for target nucleic acid.
Extensive work has been carried out to isolate and characterize DNA polymerases from many sources and for many potential uses. Antibodies to some of such polymerases have also been developed (see for example, U.S. Pat. No. 4,638,028 of Lui et al) for diagnostic tests and other potential industrial and medical uses.
Thermostable DNA polymerases have also been described, for example in WO-A-89/06691 (Cetus). These DNA polymerases have found advantageous use in PCR because of their stability at high temperatures used in certain PCR steps. Accordingly, almost everyone uses thermostable DNA polymerases when carrying out PCR. However, as noted above, the highly powerful nature of PCR has inherent problems, that is, the amplification of nonspecific nucleic acids and the formation of primer dimers. These problems are particularly acute in the presence of thermostable DNA polymerases which have some activity even at relatively lower temperatures (that is, below about 50.degree. C.).
It would be desirable to reduce or eliminate the formation of nonspecific products and primer dimers in PCR, especially with the use of thermostable DNA polymerases.
This problem has been met in one fashion as described in copending U.S. Ser. No. 880,911 (filed May 7, 1992 by Yoo, Sharkey, Christy, Jr. and Esders) by encapsulating one or more of the reagents used in PCR. The encapsulating materials are designed to melt at the temperatures normally used for PCR so the reagents are released for reaction only at the proper time.
Our colleagues have found this invention to be very useful, but there is a need for further improvement. In particular, the use of encapsulating materials can be tedious and expensive, especially in large quantities, and some PCR reagents are encapsulated only with considerable difficulty. Thus, there is a need to overcome the problems of the art without the use of encapsulation.